MAGE genes belong to the family of cancer/testis antigens. The MAGE family of genes comprises over 20 members and is made up of MAGE A, B, C and D genes (Scanlan et al, (2002) Immunol Rev. 188:22-32; Chomez et al, (2001) Cancer Res. 61(14):5544-51). They are clustered on chromosome X (Lucas et al., 1998 Cancer Res. 58.743-752; Lucas et al., 1999 Cancer Res 59:4100-4103; Lucas et al., 2000 Int J Cancer 87:55-60; Lurquin et al., 1997 Genomics 46:397-408; Muscatelli et al., 1995 Proc Natl Acad Sci USA 92:4987-4991; PoId et al., 1999 Genomics 59:161-167; Rogner et al 1995 Genomics 29:725-731), and have a yet undefined function (Ohman et al 2001 Exp Cell Res. 265(2): 185-94). The MAGE genes are highly homologous and the members of the MAGE-A family, especially, have between 60-98% homology.
The human MAGE-A3 gene is expressed in various types of tumours, including melanoma (Furuta et al. 2004 Cancer Sci. 95, 962-968.), bladder cancer, hepatocellular carcinoma (Qiu et al. 2006. Clinical Biochemistry 39, 259-266), gastric carcinoma (Honda et al. 2004 British Journal of Cancer 90, 838-843), colorectal cancer (Kim et al. 2006 World Journal of Gastroenterology 12, 5651-5657) and lung cancer (NSCLC) (Scanlan et al 2002 Immunol Rev. 188:22-32; Jang et al 2001 Cancer Res. 61, 21: 7959-7963). No expression has been observed in any normal adult tissues with the only exception of testicular germ cells or placenta (Haas et al. 1988 Am J Reprod Immunol Microbial 18:47-51; Takahashi et al. 1995 Cancer Res 55:3478-382).
Antigen-Specific Cancer Immunotherapeutics (ASCI) represent a novel class of medicines designed to train the immune system to recognize and eliminate cancer cells in a highly specific manner. As such, ASCI allow targeted treatment. ASCI have two principal components: “tumor antigens” to direct the immune response specifically against the cancer cell and “adjuvant systems” that comprise immuno-stimulation compounds selected to increase the anti-tumour immune response. MAGE-A3 antigen and constructs suitable for use in ASCI are described in WO99/40188 and encouraging phase II study results with MAGE-A3 ASCI in patients with Non Small Lung Cancer (NSCLC) have been reported recently (J. Clin. Oncol. Vol. 25, No. 18S (June 20 Suppl.) 2007: 7554).
It is important to have quantitative high throughput assays capable of specifically identifying MAGE-A3-expressing patients that would benefit from immunotherapy, monitoring MAGE-A3 expression for dosage purpose, identifying Mage-A3 expressing samples in clinical trials, or simply identify at an early stage patients with cancer. A number of applicable diagnostic methods have been described and include: Semi-quantitative RT-PCR (De Plaen et al. 1994 Immunogenetics 40(5):360-9), other PCR based techniques and low-density microarrays (Zammatteo et al. 2002 Clinical Chemistry 48(1) 25-34). Further, an improved RT-PCR method for use in conjunction with MAGE-A3 ASCI has been discussed in WO2007/147876.
The greatest disadvantage of the existing assays is that they require RNA isolation to assess MAGEA3 expression. Formalin-Fixed, Paraffin-Embedded (FFPE) tumour tissue is the usual method of tumour tissue preservation within clinical centres. The fixation in formalin changes the structure of molecules of RNA within the tissue, causing cross linking and also partial degradation. The partial degradation leads to the creation of smaller pieces of RNA of between 100-300 base pairs. These structural changes to the RNA limit the use of RNA extracted from FFPE tissue to measure MAGEA3 expression levels.
An object of the present invention is to provide an improved assay that eliminates the disadvantages of the existing assays.
Gene methylation is an important regulator of gene expression. In particular, methylation at cytosine residues found in CpG di-nucleotide pairs in the promoter region of specific genes can contribute to many disease conditions through down regulation of gene expression. For example, aberrant methylation of tumour suppressor genes can lead to up or down regulation of these genes and is thus associated with the presence and development of many cancers (Hoffmann et al. 2005 Biochem Cell Biol 83: 296-321). Patterns of aberrant gene methylation are often specific to the tissue of origin. Accordingly, detection of the methylation status of specific genes may be of prognostic and diagnostic utility and can be used to both determine the relative stage of a disease and also to predict response to certain types of therapy (Laird. 2003 Nat Rev Cancer 3: 253-266).
Methylation-Specific PCR (MSP) with visualization of the results on a gel (gel-based MSP assay) is widely used to determine epigenetic silencing of genes (Esteller M et al. Cancer Res 2001; 61:3225-9.), although quantitative tests using other technologies have been developed (Laird P W., Nat Rev Cancer 2003; 3:253-66; Eads et al. Nucleic Acids Res 2000; 28:E32; Mikeska T, et al. J Mol Diagn 2007).
A number of fluorescence based technologies are available for real-time monitoring of nucleic acid amplification reactions. One such technology is described in U.S. Pat. No. 6,090,552 and EP 0912597 and is commercially known as Amplifluor®. This method is also suitable for end-point monitoring of nucleic acid amplification reactions. Vlassenbroeck et al. (Vlassenbroeck et al., 2008. Journal of Molec. Diagn., V10, No. 4) further describes a standardized direct, real-time MSP assay with use of the Amplifluor® technology.